Collagen is a natural body material useful in a wide range of medical applications. The incorporation of glycosaminoglycans (GAG) into collagen is recognized as providing for a matrix that allows for regeneration of primary tissues. Thus, collagen/GAG matrices represent a particularly useful family of collagen-containing materials.
Collagen/GAG matrices and methods for their production are described in U.S. Pat. No. 4,060,081, U.S. Pat. No. 4,280,954, and U.S. Pat. No. 4,505,266. These biodegradable matrices are useful in a variety of biochemical applications, including, but not limited to dermal replacement constructs. For example, the dermal replacement layer of the INTEGRA® Dermal Regeneration Template™ is comprised of a porous matrix of fibers of cross-linked bovine tendon collagen and the glycosaminoglycan chondroitin-6-sulfate. This commercially available bilayer membrane system for skin replacement is useful in the treatment of deep, partial-thickness, or full-thickness thermal injury to the skin such as third-degree burns. Following application to the wound, the bilayer functions as an artificial skin that provides immediate post-excisional wound homeostasis, facilitating patient recovery and relieving metabolic stress.
However, compositions comprising collagen and GAG are sensitive to the sterilization normally applied to medical products. Thus, presently compositions comprising collagen and GAG which are to be used as matrix materials are commonly immersed in 70 percent isopropyl alcohol (IPA) for packaging and storage. Immersion in IPA does not alter either the cross-link density or other important structural features of collagen-mucopolysaccharide composites which render them useful as matrices or scaffolds.
However, under current Federal and Drug matrix materials cannot be labeled as sterile. As a result, under FDA regulations, collagen/GAG matrix materials cannot be used sub-dermally.
Further, packaging the collagen/GAG matrix materials in IPA is inconvenient for the end user because IPA must be treated as a hazardous chemical waste that must be disposed of properly. In addition the shipping costs for IPA packaged materials are substantially higher.
There is, therefore, a need for methods of sterilizing collagen/GAG compositions to be used as matrix or scaffold materials.
Various attempts to sterilize collagen alone and collagenous tissues have been made.
Physical sterilization methods include heating by boiling, autoclaving and/or microwave. However, heating results in coagulation of collagen containing soft tissues. Further, temperatures above 60° C. to 65° C. result in denaturing of collagen.
Chemical sterilization methods include exposure to agents such as formaldehyde and glutaraldehyde. However, these agents also cross-link collagen, thereby increasing its stiffness, while decreasing its remodeling ability following implantation (Kato et al. Journal Biol. Jt. Surgery 1991 73:561). These chemical sterilization methods also leave residual amounts of the chemicals in the collagen. Accordingly, chemical sterilization methods are not applicable to terminal sterilization since materials with chemical residuals cannot be implanted in the body. Further, chemical sterilants such as ethyl and isopropyl alcohol are not suitable for collagen sterilization as these agents are not sporicidal. The chemical sterilant ethylene oxide is also not suitable for wet aqueous materials as the hydrolysis of ethylene oxide becomes a concern.
U.S. Pat. No. 5,460,962 discloses a method for sterilizing collagen and collagenous tissues with low concentration peracetic acid solutions in either neutral or high ionic strength that prevent or minimize swelling of the collagen or collagenous tissue. However, since matrices with residual periacetic acid cannot be implanted in the body, this method is also not practical for terminal sterilization.
Gamma-irradiation between 0.5 and 2.5 mega-rads has also been used to sterilize tissues. However studies have shown that collagen is damaged by gamma-irradiation at 1 mega-rad (Chueng et al. J. Biomedical Material Research 1990 24:581–590), and at sterilizing doses it is damaged to a degree that compromises the desired function of the present matrices.
Accordingly, attempts to sterilize collagen alone have been relatively unsuccessful. Further such methods for sterilization are oftentimes not applicable to compositions comprising both collagen and GAG. For example, ethylene oxide reacts irreversibly with the free amino groups within the collagen/GAG matrix thereby altering the material chemically.
Sterilization by beta-irradiation has been suggested as a means to avoid decreases in mechanical properties observed following gamma-irradiation of collagen and chondroitin 4-,6-sulphate biomaterials designed for the coverage of serious burns (Berthod et al. Clinical Materials 1994 15(4):259–65). However, this method cannot be applied to wet collagen/GAG samples of any significant thickness which are often required in medical applications.
Electron beam sterilization processing was developed in 1956 by Johnson and Johnson for sterilization of medical devices. The original systems were inferior compared to gamma irradiation. However, more recent improvements in the reliability and performance of critical accelerator components from industrial involvement in the development of radiographic and oncology machines has resulted in the reevaluation of electron beam technology.
Phase I SBIR Grant [DK56504-01 submitted by Integra LifeSciences Corporation] which was funded on Feb. 19, 2001 suggests use of electron beam (E-beam) irradiation as an alternative to gamma-irradiation to sterilize RGD peptide-containing collagen-GAG matrices for use in islet cell transplantation. High intensity E-beam processing radiation exposure time is less than one minute as compared to 2 to 6 hours with gamma-irradiation. This time decrease is hypothesized to result in less damage from oxidative effects on the products due to the shorter time frame for free radicals to interact with oxygen molecules in and around the product producing ozone and oxidative damage. Increasing the amount of cross-linking is proposed as a possible means for accommodating for minimal degradation expected upon sterilization of the RGD peptide-containing collagen/GAG matrix by E-beam irradiation while retaining the degree of cross-linking required for the maintenance of optimal biological activity.
E-beam irradiation has also been used to sterilize small intestinal submucosa, a resorbable biomaterial containing a high content of glycosaminoglycans that is used in tissue grafts of vascular, urologic, dermatologic, neurologic and orthopedic injury (Hodde et al. Tissue Engineering 1996 2(3):209–217; Lantz et al. J. Invest. Surg. 1993 6:297–310). However, this material is not intended for use in promoting tissue regeneration nor has that use for it been demonstrated.
Studies comparing the physicochemical and biodegradative properties of human amniotic membranes cross-linked via gamma-ray or E-beam radiation or via glutaraldehyde showed a decrease in tensile strength and elongation at break of the amniotic membrane in both the gamma-ray and E-beam irradiated membranes. This decrease in tensile strength and elongation at break of the amniotic membrane are suggested to be caused by scission of collagen chains through irradiation (Valentino et al. Archives of Otolaryngology—Head and Neck Surgery 2000 126(2):215–9).
Further, as demonstrated herein, E-beam radiation of INTEGRA® Dermal Regeneration Template™ which comprises a collagen/GAG matrix caused both physical and chemical changes in the INTEGRA® Dermal Regeneration Template™. E-beam sterilization experiments in a dry collagen/GAG matrix expected to be less susceptible to radiation damage as compared to wet collagen also revealed changes in the molecular weight and cross-linking density causing biological consequences.